98 NORMAN L. LAWRENCE 



in the spores of B. terminalis, as germination was not evident in this species 

 when incubated with the specific germination nutrients above 65 °C. 



(2) Use of pH's unfavorable to germination. The use of a pH that is 

 unfavorable to the germination of a particular species may also be successful, 

 providing it is employed in instances where the pH range for germination 

 is rather acute, thereby allowing the selection of a pH which will yet be 

 feasible for some enzymatic studies (Harrell and Halvorson, 1955). 



(3) Use of substrates not conducive to germination. (Stewart and Hal- 

 vorson, 1953). Special care must be exercised where the substrate added 

 may be conducive to germination. This is of particular importance when 

 aged spores are employed to study a reaction which necessitates the addition 

 of a substrate that partially fulfills the normal germination requirement of 

 that species. As an illustration, a significant proportion of aged B. terminalis 

 spores has been observed to germinate in either L-alanine or adenosine alone, 

 although fresh spore suspensions — that is, spores harvested and stored less 

 than a week — of this species require the presence of both compounds before 

 germination is evident and complete. Several other similar illustrations were 

 mentioned in yesterday's session. Consequently, one who is concerned with 

 enzymatic studies employing dormant intact spores should be reminded to 

 check the reaction mixture to determine whether or not germination has 

 occurred during experimentation. 



(4) Use of germination inhibitors. (Murty and Halvorson, 1957a; Levin- 

 son and Sevag, 1953; Powell, 1950). In regard to the use of specific in- 

 hibitors of germination, some of those discussed in last night's session, or 

 others, may prove quite useful, providing they do not interfere with the 

 enzymatic system under consideration. 



(B) As a second criterion, enzymes classified in this category must 

 display activities under conditions where the possibility of activating dormant 

 enzymes in the intact spore is unlikely. This stipulation has become im- 

 perative with the discovery of dormant enzyme systems — so-called because 

 their activities are normally absent or undetectable in the fresh dormant 

 intact spores and require some means of activation before measurements can 

 be made (Church and Halvorson, 1956; Murty and Halvorson, 1957b). 

 It has therefore become essential that one consider this possibility of activa- 

 tion, which is referred to as ungerminated spores that display heretofore 

 undetected enzymatic behavior, as a direct result of some activating stimulus, 

 or stimuli. These physiological changes at this time appear to be somewhat 

 analogous to that occurring in spores during storage. Among the practices 

 which consequently should be avoided when studying enzymes active in the 

 dormant intact spore are: (1) heat shocking, particularly prolonged heat 



